Summary T cells play a central role in antitumor and antiviral immunity. In chronic infections and cancer, constant stimulation of the T cell receptor (TCR) often leads to exhaustion of T cells. Exhausted T cells are functionally incompetent and therefore less efficient to clear viruses and tumors. Reversing T cell exhaustion, such as with anti-PD-1 treatment, is used as therapy for a variety of cancers. However, challenges for successful immunotherapy remain, such as low response rate and immune-related adverse events. Thus, understanding how T cell exhaustion is regulated will help discover novel therapeutic targets and improve current therapies for chronic infections and cancer. We recently revealed a critical role of transmembrane protein 16 (TMEM16) F in restricting T cell exhaustion. We found that TMEM16F-KO T cells become hyperactivated during the early phase of chronic viral infection. This overactivation ultimately leads to severe T cell exhaustion and uncontrolled virus burden. Mechanistically, we found that TMEM16F is required for TCR-induced formation of multivesicular bodies (MVBs), a process that downregulates TCR signaling to prevent overactivation of T cells. Our hypothesis is that increased activity of TMEM16F could break T cell exhaustion, a novel therapeutic strategy applicable to chronic infections and cancer. We will support our hypothesis with the following aims: 1) Elucidate the role of TMEM16F in regulating T cell exhaustion in cancer. We will challenge wild-type (WT) or TMEM16F-KO mice with B16F10 melanoma cells. We will subsequently examine survival, tumor size, and responses of tumor-infiltrating T cells. To measure antigen-specific T cell responses, we will establish a tumor model using B16F10-GP33 combined with adoptive transfer of GP33-specific P14 TCR-transgenic WT or TMEM16F-KO T cells. Mechanistically, we plan to study the impact of TMEM16F on T cell receptor signaling in vivo. We will also use a genetic mouse model to investigate TMEM16F and T cell exhaustion toward autochthonous tumors. Ultimately, we intend to translate the role of TMEM16F in T cell responses to melanoma patients. 2) Examine how targeted activation of TMEM16F affects T cell exhaustion in chronic viral infection and cancer. We will establish a retrogenic mouse model to increase TMEM16F activity in vivo. To generate a T cell-specific system for overactivation of TMEM16F in vivo, we will perform lentiviral infection and subsequent adoptive T cell transfer to generate P14-chimeric mice. Based on these two mouse models, we will explore whether overactivation of TMEM16F affects the development of T cell exhaustion and the efficacy of anti-PD1 blockade in the context of LCMV chronic infection or B16F10 melanoma challenge. Overall, our proposed study will potentially identify TMEM16F as a novel target for therapies against chronic infection and cancer.